1. Field of the Invention
The present invention relates to an apparatus and a method using the apparatus for the more efficient cleaning of nucleic acids, and to a kit for carrying out this method.
2. Description of Related Art
As is known from the state of the art, the isolation of nucleic acids from complex biological starting materials is carried out under strongly denaturising and reducing conditions. The starting materials containing the nucleic acids are partially solubilised using protein-degrading enzymes, and the escaping nucleic acid fractions are cleaned by means of a phenol/chloroform extraction step. The nucleic acids can then subsequently be obtained from the aqueous phase by means of dialysis or ethanol precipitation (J. Sambrock, E. F. Fritsch and T. Maniatis, 1989, Cold Spring Harbor, “Molecular Cloning”).
The methods for the isolation of nucleic acids known from the state of the art have the disadvantage, however, that they are time-consuming and necessitate a considerable outlay on apparatus. In addition, such methods can be shown as being hazardous to health due to the chemicals used, such as phenol and chloroform.
In order to avoid the harmful and expensive phenol/chloroform extraction of nucleic acids combined with an additional reduction in the time required for carrying out experiments, various alternative methods for the isolation of nucleic acids from different biological starting materials have been developed in the past.
These include methods, which are based on a method described for the first time by Vogelstein and Gillespie (Proc. Natl. Acad. Sci. USA, 1979, 76, 615-619) for the preparative and analytical cleaning of DNA fragments from agarose gels. This method combines the dissolving of the agarose containing the DNA band to be isolated in a saturated solution of a chaotropic salt (NaJ) with a subsequent bonding of the DNA to glass particles.
The DNA fixed to the glass particles is subsequently washed with a washing solution (20 mM Tris HCl [pH 7.2], 200 mM NaCl; 2 mM EDTA; 50% v/v ethanol) and then eluted from the carrier particles. This method has been frequently modified in the course of time and is presently used for different methods of extraction and cleaning of nucleic acids of widely differing origin.
A large number of reagent systems (kits) currently exist, mainly for the cleaning of DNA fragments from agarose gels and also for the isolation of plasma DNA from bacterial lysates, which is associated with an additional isolation of longer-chain nucleic acids (genomic DNA, cellular RNA) from blood, tissue and also cell cultures.
These commercially available kits are based on the sufficiently well-known principle of bonding nucleic acids to mineral carriers under the presence of solutions of different chaotropic salts, wherein the carrier materials, for example, contain suspensions of finely ground glass powder (e.g. Glasmilk, BIO 101, La Jolla, Calif.), diatomic earths (Sigma) or even silica gels (Qiagen, DE 41 39 664 A1).
In addition, various methods for the isolation of nucleic acids have been used in the state of the art in order to bond the starting materials to a DNA-bonding solid phase by means of a chaotropic buffer (U.S. Pat. No. 5,234,809). Here, the chaotropic buffers are used both for the lysis of the starting material and also for the bonding of the nucleic acids to the solid phase.
With this method, nucleic acids from small sample quantities can be used, especially when used for the isolation of viral nucleic acids. The incubation of the starting material with the chaotropic buffer and with the DNA-bonding solid phase has the disadvantage that the cell decomposition, which is to be realised by the chaotropic buffer, cannot be used for all materials, and also can only be used extremely inefficiently and with considerable time expenditure particularly for larger quantities of starting materials. Therefore, additional mechanical homogenisation methods are often used (e.g. in the isolation of DNA from tissue samples). Varyingly high concentrations of different chaotropic buffers must be used for different objectives, and therefore—by nature—cannot be applied universally.
In order to simplify the possibly difficult lysis of the starting material, a series of commercially available products can be used for isolating the nucleic acids, which however are then no longer based on an easily manageable so-called “single tube” method.
The chaotropic salts necessary for the subsequent bonding of the nucleic acids (e.g. to centrifugation membranes) must be added to the lysis preparation in a special method step when the lysing is complete. On the other hand, these chaotropic salts cannot be part of the lysis buffer however, as the protein-destroying function is inherent to the chaotropic substances and these would also destroy the proteolytic enzymes necessary for efficient lysis.
The methods described above and known from the state of the art for isolating nucleic acids using chaotropic salts have become established worldwide and are freely applied in their millions using commercially available products.
According to the principle of starting-material lysis, in a simple execution, the nucleic acids are bonded to the solid phase of a glass or silica membrane, which is located on a carrier substance in a centrifuge column. The bonded nucleic acids are subsequently eluted with a buffer of lower ion strength.
The physical-chemical principle of the bonding of nucleic acids to mineral carriers in the presence of chaotropic salts has been explained in professional circles. It has been postulated that the bonding of nucleic acids to the surfaces of mineral carriers is based on a breaking down of superimposed structures of the aqueous environment, by means of which the nucleic acids adsorb on the surface of mineral materials, in particular of glass or silica particles.
When the concentrations of the chaotropic salts are high, the reaction proceeds almost quantitatively. For this reason, a buffer composition with high ion strengths of chaotropic salts is important for bonding nucleic acids to a nucleic-acid-bonding solid phase.
For the bonding of nucleic acids to the respective carrier surfaces, the buffer solution contains at least one chaotropic salt as its main component. Under certain circumstances, this even includes the lysis buffer or, in systems that use proteolytic enzymes, a necessary bonding buffer, which is added to the starting material after lysis is complete.
The Hofmeister series for salting out negatively charged, neutral or basic protein solutions forms the basis for chaotropic salts. Chaotropic salts are characterised in that they denature proteins, increase the solubility of unpolar substances in water, and destroy hydrophobic interactions. These characteristics also effect the destruction of superimposed structures of the aqueous environment with buffer systems of chaotropic salts, in order to promote the bonding of the nucleic acids to selected solid phases.
The best-known examples of chaotropic salts for isolating nucleic acids include sodium perchlorate, sodium iodide, potassium iodide, guanidinium-iso-thiocyanate and guanidinium hydrochloride. However, they are cost-intensive and also, to some extent, toxic or irritant.
A method for the isolation of DNA from tissue and cell lines is described in the state of the art, in which the cells are dispersed in a buffer containing guanidinium hydrochloride, and precipitated in ethanol (Analytical Biochemistry 162, 1987, 463). On the one hand, this method is susceptible to contamination, but on the other, a usable nucleic acid product can be isolated within a few hours.
In addition, a method for the isolation of nucleic acids using antichaotropic substances is known from the state of the art. Here, an improved isolation of nucleic acids can likewise be achieved by the addition of antichaotropic salts in a lysis/bonding buffer system. Antichaotropic components include ammonium, caesium, sodium and/or potassium salts, preferably ammonium chloride. When using lysis/bonding buffer systems without the chaotropic salt constituents, nucleic acids, particularly genomic DNA, can be bonded to a mineral carrier material and also eluted under the usual reaction conditions.
Furthermore, it has been found that at least the same quantitative and qualitative results are achieved with lysis/bonding buffers, the main components of which are ammonium salts, for example, instead of chaotropic salts, in extractions of genomic DNA from starting materials of different complexity (e.g. blood, tissue, plants) using the previously common (alternative) reaction components and carrier materials and with the same reaction process.
It has thus been observed that with salts, which do not denaturise but stabilise proteins, and do not degrade but reinforce hydrophobic interactions, it is also equally possible to isolate nucleic acids from complex starting materials.
Low concentrations of salts (less than 1 M) are adequate for bonding nucleic acids to solid carriers. In certain applications, concentrations less than 0.5 M are preferred, higher ion concentrations being necessary for the quantitative isolation of nucleic acids from larger quantities of starting materials.
These lysis/bonding buffer systems known from the state of the art, which have at least one antichaotropic salt component, are therefore capable of bonding nucleic acids to solid phases, which have a negatively charged surface, or which contain surfaces, which are capable of exhibiting a negative charge potential.
The reaction sequence of the isolation of nucleic acids from a complex starting material is realised by carrying out the lysis of the starting material, bonding the nucleic acids, washing the bonded nucleic acids, and eluting the nucleic acids in a reaction vessel, and requires at least one centrifugation step.
The methods of isolating plasmid DNA with the usual midi and maxi systems previously known from the state of the art are, however, only scaled-up mini preparations whose columns have either been increased to a larger diameter or supplemented by very many membrane layers in order to achieve the required capacity. Systems of this kind are currently supplied by manufacturers such as Stratagene or Sigma for example.
The glass fibre membranes employed here work almost exclusively using chaotropic salts for selective bonding to the surface of the membrane.
The main disadvantage is the complex handling (many centrifugation steps with large floor-mounted centrifuges). Added to this is a relatively large elution volume combined with a large dead volume due to the large internal surfaces and the resulting larger membrane volumes.
As a result of this surface area, which is necessary for increasing the capacity, the processing of larger preparations with an apparatus based on the mini format while maintaining a chaotropic bonding chemistry has not previously been possible.
On the other hand, plasmid isolation based on an alcoholic bonding chemistry has previously only been possible using the Invisorb® Plasmid Kits produced by the company Invitek (Berlin). But even in these kits, columns are used, which are not suitable for preparations on a midi/maxi scale due to their dimensions. The disadvantages already described therefore apply to these.